Vol. 140, No. 1

JOURNAL OF BACTERIOLOGY, Oct. 1979, p. 229-239 0021-9193/79/10-0229/1 1$02.00/0

Escherichia coli Pleiotropic Mutant That Reduces Amounts of Several Periplasmic and Outer Membrane Proteins BARRY L. WANNER,t APARNA SARTHY, AND JON BECKWITH* Department of Microbbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115

Received for publication 18 June 1979

We have isolated a mutant of Escherichia coli K-12 that is reduced from 6- to 10-fold in the amount of alkaline phosphatase found in the periplasmic space. The reduced synthesis is not due to effects at the level of transcription regulation of the phoA gene, the structural gene for the enzyme. In addition, the mutation (termed perA) responsible for this phenotype results in reduced amounts of possibly six or more other periplasmic proteins and at least three outer membrane proteins. One of the outer membrane proteins affected is protein la (D. L. Diedrich, A. 0. Summers, and C. A. Schnaitman, J. Bacteriol. 131:598-607, 1977). Although other possibilities exist, one explanation for the phenotype of the perA mutation is that it affects the cell's secretory apparatus.

The secretion across cellular membranes of a number of proteins appears to proceed by a mechanism proposed in the signal hypothesis (6). According to this hypothesis, secreted proteins are synthesized initially as precursors with an amino-terminal sequence of 15 to 30 amino acids (the signal sequence); these sequences serve to attract polysomes engaged in their synthesis to the membrane. These proteins are then discharged vectorially across the membrane during protein synthesis, and either during or after this process the signal sequence is cleaved. Evidence has accumulated for the existence of signal sequences for a large number of secretory proteins and certain membrane proteins in eucaryotic systems (14). In the gram-negative bacterium Escherichia coli, export of a number of envelope proteins (including inner and outer membrane and periplasmic proteins) appears to follow the same mechanism. Signal sequences have been found in the case of several periplasmic proteins and outer membrane proteins and for one cytoplasmic inner membrane protein which is transmembranal (for review, see ref. 4). Certain of these proteins have been shown to be synthesized preferentially on membrane-bound polysomes and to be discharged across the membrane cotranslationally (36). Finally, mutations have been characterized which alter the signal sequence of three different envelope proteins and which, as a result, prevent either secretion or processing of these proteins (2, 12, 19). Although the broad outlines of the mechanism t Present address: Department of' Biology, Massachusetts Institute of Technology, Cambridge, MA 021:39.

of secretion appear established, the details of how proteins actually traverse membranes and what the role of processing is remain obscure. For instance, little is known about the role of ribosomes or translation factors in the interaction between polysomes and the membrane and in the energetics of secretion. Whereas certain proteins (ribophorins) have been identified in rough endoplasmic reticulum or eucaryotic cells as playing a possible role in ribosome binding (16, 39), no similar evidence exists in bacteria. We believe that an essential component of studies on the process of secretion is the isolation of mutants which alter the secretion process. In particular, mutant strains defective in the secretion of classes of envelope proteins are likely to be altered in some component of the export machinery, e.g., a ribosomal protein, membrane protein, or processing enzyme. These mutants, in turn, would provide a handle on detecting the altered component and would permit assigning it a role in the process. One approach we are taking to this problem is to isolate mutants defective in the secretion of the E. coli enzyme alkaline phosphatase, which is localized in the periplasmic space (21). Alkaline phosphatase, the product of the phoA gene, is synthesized at high levels under conditions of phosphate starvation. Its synthesis is under the control of at least two regulatory genes, the phoB gene, which is a positive control factor, and the phoR gene, which is a negative control factor (7, 13). How these two regulatory proteins interact is still not understood. Alkaline phosphatase is made initially on membrane-bound 229

230

WANNER, SART'HY, AND BECKWITH

polysomes (3) as a larger precursor (15) and is discharged vectorially across the cytoplasmic membrane during its synthesis (36). A partial amino-terminal signal sequence for this enzyme has been determined (Sarthy, Fowler, Zabin, and Beckwith, unpublished data). Processing of the alkaline phosphatase precursor has been accomplished in vitro with both outer (15) and inner membrane preparations (Inouye and Chang, unpublished data). In this paper we describe a method whereby we can detect mutations unlinked to the phoA gene which reduce synthesis of alkaline phosphatase. This is done by mutagenizing the entire E. coli chromosome of a phoA-negative strain and then introducing an unmutagenized phoA gene carried by a specialized transducing phage. Several mutants have been isolated in this way. We describe one which may be defective in the export of alkaline phosphatase and other envelope proteins.

.J. BACTERIOL.

MATERIALS AND METHODS Media. Minimal medium M63 was used routinely (22). MOPS medium was used for all minimal medium liquid cultures (24). LB andl sugar tetrazolium plates have been described previously (22). Plates containing 0.2'7. /3-glycerol phosphate (filter sterilized) were based on MOPS medium. The indicator plates used for mutant isolation were based on MOPS medium and in addition contained 1.5% agar, 2.5 x 10 :' 2,3,5-triphenyl tetrazolium chloride, 2.5 x 10 M potassium phosphate, 0.125', tryptone (Difco), 0.2% lactose, I0t M isopropyl-/3-D-thiogalactopyranoside (Sigma), and 2 x 10 ;'i 5-bromo-4-chloro-3-indolyl-phosphate (XP) (Bachem). Because these plates are buffered and tetrazolium acts as a redox indicator, Lac' colonies turn red and Lac colonies remain white. These plates were adopted from a medium described by Lin et al. (18). XP produces a blue dye when hydrolyzed by alkaline phosphatase (7). XP plates were based on MOPS medium and in addition contained 1.5% agar, 10 4 M potassium phosphate, 0.2' glucose, and 2 x 10 XP. Bacterial strains. The genotypes and origins of bacterial strains are listed in I'able 1. All are E. coli

TABLE 1. Bacterial strains" Strain

CA7087 AB2847 107010 107011 C-4 (DPh3) CS397 EC1, 2, 3, 6 through 10 XPh4 XPh24

Genotype

XPh31 XPh37 BW440 BW442

HfrH proC thi aroB351 malT354 txs-354 supE42 aroB his-29 trpA9605 metE i/cn trpR bgl+ HfrC phoT thi ompB156 aroB351 tsx supE42 Hfr lac+ f(proB-lac)xui, lacZ524 phoR phoA20 rpsL lacZ524 trp rpsL A(brnQ-phoA-proCphoB,R) lacZ524 rpsL A (brn Q phoA pr oC phoB,R) XPh31 lysogenic for ,80d 17 malT derivative of XPh4 lacZ524 phoR phoA20 rpsL aroB

BW479.48; .57

rpsL aroB phoR; phoR+

BW489.1; 2

rpsL phoR perA+; perA

BW490.9; 12

rpsL perA; perA'

BW495 BW496

rpsL phoR aroB nmalT rpsL phoR aroB perA

BW497

rpsL phoR aroB ompB

BW498 BW499

malT derivative of BW489.1

rp.sL phoR ompB

BW500.1 rp.sL phoR perA BW500.10 rpsL phoR ompB BW500.61 rpsL phoR BW503.1 rpsL phoR ompB BW503.2 rpsL phoR perA BW503. 10 rpsL phoR BW503.230, 420, 425 rpsL phoR perA ompB " Abbreviations used are those of Bachmann et al. (1).

Soturce or reference F. Jacob E. coli Genetic Stock Center E. Brickman L. Guarente A. M. Torriani A. Pugsley (5) (7) (7)

Trp+ transductant of XPh24 See text Mal+ transductant of BW440 with P1 grown on 107010 Lac+ transductant of BW442 with P1 grown on 107010 Aro+ transductant of BW479.48 with P1 grown on mutant 2 Aro+ transductant of BW479.57 with P1 grown on mutant 2 See text Mal+ transductant of BW495 with P1 grown on BW489.1 Mal transductant of BW495 with P1 grown on CS397 See text Mal+ transductant of BW498 with P1 grown on CS397 See T'able 7 See Table 7 See Table 7 See Table 8 See Table 8 See Table 8 See Table 8

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MUTATION AFFECTING SECRET'ED PROTEINS

K-12 derivatives. malT derivatives were selected as Xvir-resistant Mal- colonies on maltose tetrazolium plates. Bacteriophage P1, grown on strain 107010, was used to transduce malT derivatives to Mal' aroB. This lysate was also used to transduce BW442 to Lac'. Lac+ transductants were purified and tested for their PhoA and PhoR phenotypes. Bacteriophage P1 grown on mutant 2 (see below) was used to transduce aroB derivatives to prototrophy or malT derivatives to Mal' to introduce the perA mutation, which was approximately 60% linked to each of these loci. Bacteriophage P1 grown on CS397 was used to transduce malT derivatives to Mal+ to construct otherwise isogenic ompB derivatives. The EC strains are Hfr strains which were constructed by integration of an F'lac into different regions of the chromosome (5). As a result, these strains have various points of origins and different directions of transfer in matings with F- strains. Phages. The following p80 specialized transducing phages were isolated in this laboratory (7): 080dl3 phoB +proC+, 080d161acZ+ Y+proC+phoA +, 480d17proC+phoA+, and 080plac+ (31). The Xp(phoA-lacZ)428 fusion-carrying phage was isolated by the method of Casadaban (9; A. Sarthy and J. Beckwith, unpublished data). The 4>80plphoA+ phage was isolated after UV induction of a tandem 080,080d16 lysogen. In such a lysogen the phoA gene is transposed to near the helper phage. Aberrant excision events can occur which permit the phoA + gene to become excised with the helper phage. Such plaque-forming phoA+ phage were detected as blue plaques when a portion of the lysate was grown on a phoA strain on XP plates. 480pphoA + phage were found at a frequency of about 10-4 per phage particle. Isolation of promoter-like phoA mutations. Strain XPh37, which has a phoB deletion, carries a defective 80phoA + phage transposed to the att8O site. The phoB gene is deleted so that phoA is expressed at very low levels (7). We attempted to isolate a trpphoA fusion. Spontaneous tonB deletions were sought which moved the phoA gene closer to the trp operon. After UV mutagenesis of a tonB-trp deletion derivative, survivors were spread onto XP plates. A few dark-blue colonies were found. Such colonies had a high constitutive level expression of alkaline phosphatase. In none of these strains was alkaline phosphatase under the control of tryptophan. Instead, it appears that these mutations had altered the phoA promoter so that it was independent of the phoB gene product. The mutations were designated pho(Bin) for phoBindependent expression of the phoA gene. It was reasoned that if the pho(Bin) mutation were a promoter-like mutation it would be possible to recombine the mutation onto the plaque-forming phoA + phage. This was done by UV-irradiating the host (to increase recombination) and by infecting with the 48OplphoA + phage at a multiplicity of infection of 5. 48OplphoA + phage which incorporated the pho(Bin) mutation were detected as blue plaques when a portion of the lysate was grown on a phoA phoB double mutant on XP plates. 480pho-1003(Bin)phoA+ phage were found at a frequency of about i0' per phage particle.

231

Genetics. Bacteriophage P1 transductions, bacterial matings, and other techniques used in molecular genetics were performed as described previously (22). Mutagenesis with N-methyl-nitrosoguanidine was performed as described previously (22). A concentration and time were chosen which gave 50% survival of the mutagenized bacteria. Generally, 0.1 to 1% of the survivors became Mal- as indicated on maltose tetrazolium plates. Enzyme assays. Alkaline phosphatase and /3-galactosidase assays were performed as described previously (7, 22), except all cells were treated with sodium dodecyl sulfate (SDS) and chloroform before assay (38). Cells were harvested and washed with MOPS medium lacking a carbon and phosphate source before preparation for assay. Cells were separated from the assay mixture after incubation by centrifugation. RNase I activity was assayed using '4C-uniformly labeled polyadenylic acid (Miles) as described previously (40). Assay tubes contained between 1 and 10 ,Lg of protein. The assay was linear for 1 h when less than 40% of the radioactivity was solubilized. '4C-Polyadenylic acid, 2 x 10-2 pCi (0.1 IiCi/pmol of P), was added to each tube. Units represent the percentage of 14C polyadenylic acid solubilized and are per milligram of protein. Less than 30% of the substrate was hydrolyzed in a particular assay. Cyclic phosphodiesterase and nonspecific acid phosphatase activities in the osmotic shock fluids were assayed as described previously (1 ). Cell fractionation. Osmotic shock fluids were prepared from stationary-phase cultures grown in LB medium essentially as described previously (25), except the cells were suspended in ice-cold 0.5 mM MgCl2 after the EDTA-sucrose stage. Viable counts were routinely performed on all cultures before and after shocking. All data presented were from cultures in which at least 70% of the total alkaline phosphatase was released into the cold osmotic shock fluid without any detectable loss of cell viability. About 1% of the induced ,B-galactosidase activity was simultaneously released. The membrane fractions were prepared as described previously (10). The cells were harvested during the logarithmic growth phase in glucose-MOPS medium at an optical density at 410 nm of about 2.0. An optical density at 410 nm of 1.0 corresponds to 3 x 108 to 3.5 x 108 cells per ml.

Immunoprecipitation of alkaline phosphatase. Rabbit anti-alkaline phosphatase serum was a kind gift of Annamaria Torriani. The immunoglobulin G fraction was partially purified on Sephadex G100 before use. SaCI reagent was a kind gift of Gary Siebert. Tubes containing the cell extract and 2 mg of bovine serum albumin per ml in 0.01 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.4) containing 2% Triton X-100 were centrifuged at 10,000 x g for 5 min. Immunoprecipitation was promoted with SaCI reagent as previously described (32). Polyacrylamide gel electrophoresis. All polyacrylamide gels were prepared in slabs 0.75 mm thick and 10 to 30 cm long. The Tris-glycine buffer system of Laemmli (17) was used. A stacking gel was used in all experiments, and it contained 5.0% acrylamide, 0.1367% bisacrylamide, and 0.1% SDS.

232

WNANNER, SARITHY, ANI) BECKNNWIT1 H

Protein assay. T'he protein concentration in cell extracts was determined colorimetrically by the method of Lowry et al. (20). Screening for mutants. We designed a procedure for the isolation of mutants unable to secrete periplasmic proteins. This procedure is based upon the assumption that some factor(s) exists which is necessary for the secretion of periplasmic proteins, e.g., alkaline phosphatase, but which is not necessary for the synthesis of cytoplasmic proteins, e.g., /3-galactosidase. Mutants are detected on agar medium which includes indicators for both alkaline phosphatase and /3-galactosidase synthesis. In addition, since mutations defective in the secretion of a number of envelope proteins may be lethal to the cell, we have adopted the general approach used by Schedl and Primakoff (30) for the detection of conditional lethal mutants affecting tRNA synthesis. The medium used contains a tetrazolium dye, an indicator for lactose metabolism, and XP, an indicator for alkaline phosphatase. This medium was initially developed by Brickman and Beckwith for a different purpose. On this medium, PhoA- LacZ colonies are white, PhoA LacZ' colonies turn red, PhoA' LacZ colonies turn blue, and PhoA' LacZ+ colonies turn purple. E. coli strain XPh4, which is lacZphoA phoR rpsL (strA), was mutagenized with nitrosoguanidine and then cultured in tryptone yeast extract mediunm for 4 h at 30°C. Diluted portions were spread on indicator plates to yield about 300 colonies per plate. The plates were incubated at 30°C until the colonies grew to I to 2 mm in diameter (approximately 36 h) to allow any temperature-sensitive mutants to grow, after which the plates were incubated at 42'C. One hour later each plate was sprayed with 1 ml of a prewarmed phage miixture containing 2 x 10"' S00pphoA+ and 2 x 10"' (80placZ+ phage. Incubation was continued at 42°C. Sprayed colonies which exhibited a red color were picked and purified for further study. These mutants were presumed to be defective in alkaline phosphatase but not /3-galactosidase synthesis. Isolation of perA revertants. PhoA+ revertants of the perA strain were isolated by selecting for growth on MOPS plates containing 0.2w /3-glycerol phosphate as the sole carbon source. Onlv those strains producing high levels of alkaline phosphatase will be able to hydrolyze the 8(-glycerol phosphatase to glycerol and thus use glycerol as a carbon source.

RESULTS Isolation of mutants. Mutants were isolated which were defective in alkaline phosphatase but not /3-galactosidase synthesis, as described in Materials and Methods. These mutations are unlikely to be in the phoA gene, since the chromosome but not the phoA gene was mutagenized. After further characterization, out of approximately 16,000 colonies, 4 mutants were found which exhibited an alkaline phosphatasenegative phenotype when infected with the 80pphoA+ phage. To further verify the presumed phenotype of these strains, each was lysogenized with a qp80 transducing phage (080d16,

eJ.

BACT'IERIOLI.

ref. 7), which carries lacZ+ and phoA+. Representative results from enzyme assays of lysogens of the parental and two mutant strains are shown in Table 2. The results with mutants 3 and 4 were identical to those with mutant 1. In the case of all four mutants, alkaline phosphatase but not /8-galactosidase synthesis was affected. Characterization of mutants as regulatory and nonregulatory types. Mutations within the phoB gene, which codes for a positive effector for phoA gene expression, cause loss of expression of the phoA gene. It seemed likely that some mutants contained mutations in the phoB gene. A high level of expression of the phoA gene is required for growth on /3-glycerol phosphate as a sole carbon source. To test for the presence of a phoB mutation, each mutant lysogenic for a (80phoA+ phage was infected with a -80phoB + phage and transductants were selected on /-glycerol phosphate plates. Mutants 1, 3, and 4 yielded hundreds of transductants able to grow on ,B-glycerol phosphate as a sole carbon source. With the same lysate, mutant 2 did not yield any transductants. Thus, three of the four mutants are complemented by the presence of a wild-type phoB gene. The presence of phoB mutations in mutants 1, 3, and 4 was confirmed by using bacteriophage PI grown on the mutants. Lysates 1, 3, and 4 could simultaneously transduce a proC strain to prototrophy and to phoB. (The proC and phoB loci are 5074 linked by bacteriophage P1 transduction.) On the other hand, mutant 2 was shown to contain a wild-type phoB allele by such a transduction. T'ABILE 2. Assay of /3-galactosidase and alkaline phosphatase in vild-tvpe anid mutant bacteriaa" Strla, .a-(Gialacto- Alkaline phos-

sidase

p)hatase'

XPh4 (680d16)' 640 190 Mutant 1 (0S0d16) t).62 670) Mutant 2 (f80d16) 73() 28 1'he strains were grown overnight at 37°C in MOlRS medium containing 2.5 mnM glucose. 2 nrM potassium phosphate, 2 x 10 ` M ZnCI2, and 1 mM isopropvl-/3-D-thiogalactopy ranioside. Portions were diluted and plated onto indicator plates to determine the proportion of nonlysogens, which was less than 1'%. Portions were assayed as described in the text. Ut1nits are nanomoles of o-nitrophenol produced per minute per milligrami of protein at 280C.. The X,.2. of 4.5 x 10' was used to calculate all data shown. tUnits are nianomoles of p-nitrophenol produced per nminute per milligram of protein at 370C. The F,,, of 1.62 x 1()4 was used to calculate all data shown. d Mutants 1 and 2 are derived from XPh4. All three strains were made lysogenic for ¢>80d16, which carries the la(Z' ain(d phoA' genies aiand a (-80) helper phage.

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MUTATION AFFECTING SECRETED PROTEINS

To determine whether the regulation of alkaline phosphatase was altered in mutant 2, a phoR+ derivative was constructed using bacteriophage P1 grown on a proC strain. Lac' transductants were selected. (Glycyl-proline was used as a proline source to prevent catabolism of proline [38]). A few percent of the Lac' transductants became proC and also phoR +. During phosphate deprivation of this strain, alkaline phosphatase is controlled normally but is made at a rate about 8- to 10-fold below that of a related wild-type strain (data not shown). It seemed possible that the mutation in mutant 2 might be in another gene involved in the regulation of transcription of the phoA gene. We have tested this possibility in two ways. First, there exists a mutation, pho-1003(Bin), tightly linked to the phoA structural gene, which renders the gene largely independent of its normal regulation (see Materials and Methods). This mutation does not alter the cellular location of alkaline phosphatase (data not shown). The pho-1003(Bin) mutation may be a promoter mutation analogous to those in the lac promoter which cause the lac operon to be independent of a positive control factor (33), or they may be mutations or insertions which generate a new promoter. In any case, if the mutation in strain 2 were a regulatory mutation, we would expect the pho-1003(Bin) mutation to restore normal synthesis of alkaline phosphatase. Accordingly, a 080 transducing phage was used to introduce pho-1003(Bin)phoA+ gene into each of the mutant strains. Whereas a phoB mutation causes a several-hundred-fold reduction in expression from a wild-type phoA gene, it causes only a threefold reduction in the expression of the phoA + gene in a strain carrying pho-1003(Bin) (see lines 1, 2, and 4 in Table 3). On the other hand, the mutation in mutant 2 causes a quantitiatively similar reduction in phoA gene expression which is independent of the promoter-regulatory region (compare lines 1 and 2 in Table 3). Comparison ofphoA + expression in the strain carrying pho-1003(Bin) in lines 4 and 5 in Table 3 demonstrates a quantitatively similar reduction. The inability of the pho-1003(Bin) mutation, which renders phoA gene expression constitutive, to overcome the defect in mutant 2 further supports the hypothesis that mutant 2 does not contain a regulatory mutation. A second way in which we have tested whether the mutation in mutant 2 is affecting regulation of the phoA gene is by use of operon fusions in which the lacZ gene is under the control of the phoA promoter and regulatory genes. In these fusions, which were isolated by the Casadaban technique (9), the synthesis of /3-galactosidase is regulated by the phosphate

233

concentration in the medium (Sarthy and Beckwith, unpublished data), as is alkaline phosphatase in wild-type E. coli strains. A transducing phage carrying a phoA-lacZ operon fusion was used to construct lysogens of the parental and mutant strains. The data from assays of fl-galactosidase in these lysogens are shown in Table 4. Whereas the level of 83-galactosidase in the fusion strain is decreased by a phoB mutation (mutant 1), the defect in mutant 2 has no effect on the expression of /8-galactosidase when controlled by the phoA promoter-regulatory region. When two different phoA-lacZ fusions were isolated and introduced into a phoR + derivative of mutant 2 by bacteriophage P1 transduction, a similar level of /1-galactosidase was synthesized during phosphate deprivation as a related wildtype strain carrying the same fusion (data not shown). Based upon the results presented in this section, mutants 1, 3, and 4 were classified as regulatory, and mutant 2 was classified as a nonregulatory-type mutant. Furthermore, the results obtained using phoA-lacZ fusions make it likely that a similar level of phoA mRNA is transcribed in the wild type and in mutant 2. Thus, mutant 2 has some defect at a posttranscription initiation step in phoA gene expression. Synthesis of periplasmic proteins in muTABLE 3. Effect of the pho-1003 (Bin)phoA+ allele on the expression of alkaline phosphatase Alkaline phosphatase unitsb Strain'

phoA+

pho1003(Bin) phoA'

220 220 XPh4 74 0.68 Mutant 1 31 31 Mutant 2 74 0.62 XPh4 phoB 9.3 0.31 Mutant 2 phoB The indicated strain was made lysogenic for 480p phoA+ or q480ppho-1003(Bin)phoA+. " After overnight growth, each culture was harvested and assayed for alkaline phosphatase activity as described in Table 2. Units are as in Table 2.

TABLE 4. Assay of 8-galactosidase in carrying a phoA-lacZ fusion"

lysogens

f3-Galactosidaseb Strain 1,040, 980 XPh4 (Xp[phoA-lacZ]428) 27, 33 Mutant 1 (Xp[phoA-lacZ]428) 910, 1,160 Mutant 2 (Xp[phoA-lacZ]428) "After overnight growth, each culture was harvested and assayed for ,8-galactosidase activity as described in Table 2. bThe two values shown are assays of independent lysogens. Units are as in Table 2 except that they are calculated per milligram of protein.

234

WVANNER, SAR{THY, ANI) BECKWITH

J. BATE('l IOL,.

tant 2. Since mutant 2 appears to be defective electrophoresis on SDS-polyacrylamide gels. Gel in a posttranscription step in phoA gene expres- electrophoretic analysis of the parental and musion, it is a candidate for a mutant affecting tant strains lysogenic for the 080dlac+secretion of alkaline phosphatase. Such a mu- proC+phoA + transducing phage revealed that at tant might also be expected to alter the secretion least five to seven periplasmic proteins were of other periplasmic proteins. Cold osmotic markedly reduced in the cold osmotic shock shocking of cells preferentially releases those fluid in mutant 2. Spontaneous revertants of proteins believed to be located within the peri- mutant 2 were isolated. In four independent plasmic space (25), representing approximately spontaneous revertant strains, the gel pattern 10% of the cellular protein. We first tested for was similar to that of the parental strain (data the effect of this mutation on periplasmic pro- not shown). Thus, a single mutation is responteins by assaying certain enzymatic activities in sible for the observed pleiotropy. Since several shock fluids. The results from assays of alkaline periplasmic proteins were affected, the mutation phosphatase and RNase I specific activities in was designated perA. For a typical gel pattern the shock fluids are shown in Table 5. Whereas of the cold osmotic shock fluids from perA + and cold osmotic shocking of the parental strain perA strains see Fig. 1, which is discussed below. caused a release of 10%4 of the cellular protein, Gel electrophoretic analysis of whole-cell exsimilar treatment of the mutant 2 strain caused tracts of the perA ' and perA strains using seva release of about 6C% of the cellular protein. In eral one-dimensional gel systems revealed no both cases, greater than 70% of the alkaline differences in the gel pattern for cytoplasmic phosphatase was recovered in the shock fluid. proteins (data not shown). The only changes Consequently, the specific activity for alkaline seen on whole-cell gels corresponded to known phosphatase in the shock fluid from wild-type changes in outer membrane proteins and pericells is 10-fold higher in the shock fluid (units plasmic proteins. The total amount of alkaline phosphatase proper milligram of protein released) than in wildtype whole-cell extracts (units per milligram of tein in whole cells was determined by labeling total protein), and the specific activity in the wild-type and perA mutant cells with a mixture shock fluid from mutant cells is almost 20-fold of radioactive amino acids. After a 5-min labeling higher in the shock fluid than in mutant whole- period during exponential growth, the cells were cell extracts. We believe this observation ex- harvested and disrupted in a French press. Rabplains the apparent increase in the specific activ- bit anti-alkaline phosphatase serum was used to ity for RNase I in the shock fluid of mutant 2. A complex alkaline phosphatase monomers and similar increase was found for two other pern- dimers. These complexes were pelleted using plasmic proteins, namely, cyclic phosphodiester- Staphylococcus aureus Cowan 1 strain (see Maase and nonspecific acid phosphatase (data ilot terials and Methods). Autoradiography of the precipitates, which were resolved on SDS-polyshown). Portions of the shock fluids were subjected to acrylamide gels, demonstrated that the total alkaline phosphatase protein was dramatically reduced by the perA allele (see Fig. 2). No TABLE' 5. Assay of alkaline phosphatase aIn(l evidence for the presence of a precursor form of RNase I in cold osmnotic shock fluids" alkaline phosphatase was found in the inner membrane from either strain in this experiment. Alkaline Strain' phospha- RNase I' Mapping the perA mutation. For the purtase' pose of further strain construction, we mapped 120 86 (70) the perA gene. Bacteriophage P1 transductions Xph4 phoA+ 21 (80) 220 Mutant 2 phoA' demonstrated that the perA mutation was unMutanit 2 phoA+ revertant 99 (7() 10( linked to proC or il, two genes which are known to be linlked to genes affecting expression of " Cultures were grown overnight in LB me(liumi at .37°C. 'IThe cells were harvested, and the osmotic shock alkaline phosphatase as well as other phosphatefluid was prepared according to the text. regulated proteins. Matings were performed us" The phoAt gene was introduced on a ¢(80pphoA+ ing a phoA+ derivative of the original perA phage. strain as recipient and Hfr's with several differ'T'he alkaline phosphatase activity was measured ent points of origin and different orientations as in the whole cells and the shock fluid. The data shown correspond to the activity in the shock fluid per mil- donors (4). The results showed that the perA ligramr of protein released. 'I'he numbers inl parenthe- gene was located near malT at 74 min on the E. ses correspond to the percent of the total activity that coli chromosome. Bacteriophage P1 transduction confirmed these data. The data using aperA was recovered in shock fluid. 'The RNase I activitv was measured in the shock strain as a donor in a bacteriophage Pt transfluid. Portions were assaYe(l according to the text. duction are shown in Table 6. These results

VOL. 140, 1979

.!,:x'.

*.nbjpL;oyi

MUTATION AFFECTING SECRETEI) PROTEINS

235

perA ompB double mutants to the extreme difficulty in maintaining such mutants. The perA ompB mutants readily revert independently to p

.-

...0A..'

*

FIG. 1. Pattern of proteins present in the cold osmotic shock fluid from perA+ and perA bacteria. Cultures were grown overnight in LB medium, after which the cold osmotic shock fluid was extracted according to the text. A portion of the cold osmotic shock fluid equiualent to 10'0 cells was subjected to electrophoresis. The separating gel was 20 cm long and contained 12.5% acrylamide, 0.16%c bisacrylamide, and 0.1% SDS. Electrophoresis was performed according to the text. The gel was stained with Coomassie blue. Lane a, BW490.12 (perA+ phoR+); lane b, BW490.0 (perA phoR+); lane d, BW489.2 (perA phoR); and lane e, B W489.1 (perA + phoR). In lane c, 2 fig of alkaline phosphatase (AP) was used as a marker. The arrows on the left indicate the bands affected by the perA allele. The arrows on the right indicate the bands affected by the phoR allele.

FIG. 2. E. coli BW489.1 and BW489.2 were grown and labeled in glucose-MOPS medium. The cultures were harvested, and the total alkaline phosphatase protein was precipitated (see text). The precipitates were subjected to gel electrophoresis using a 20-cm gel containing 8.5% acrylamide, 0.26%7o bisacrylamide, and 0.1% SDS. An autoradiogram was made by exposing the dried gel to Kodak No-Screen film. In lanes a and d an equivalent amount of radioactively labeled total cell extract was immune precipitated from the perA + and perA strains, respectively. In lanes b and e each extract was diluted 10-fold before immune precipitation for the perA + and perA strains, respectiuely. In lanes c and f, the inner membranes were isolated and subjected to immune precipitation for the perA + and perA strains, respectively.

TABLE 6.

demonstrate that the perA gene is between aroB and malT and that it is about 60 to 70% linked to each of these loci. Preliminary evidence indicated that outer membrane protein Ia was decreased in the perA mutant (see below). The ompB locus (29), which is involved in the synthesis of two outer membrane proteins (Ia and Ib), has been mapped within the same region of the chromosome as the perA locus. The ompB gene may be a regulatory locus. The appropriate strains were constructed and used in bacteriophage P1 transductions to map the perA locus with respect to the ompB locus. The results of reciprocal transductions are given in Tables 7 and 8. By the number of wild-type recombinants obtained, we have determined that the perA and ompB loci are 97 to 98% linked. We attribute the low recovery of

Bacteriophage P1 transduction of the perA allelea

Unselected markerb

mal+ 2

mal

79 perA+ 80 54 perA Recipient, malT perA+ aroB; donor, malT+ perA aroB+; selected, aro+ (215). E. coli AB2847 was transduced to prototrophy with a bacteriophage P1 lysate grown on mutant 2. Aro+ transductants were selected on glucose M63 minimal plates. bAfter two consecutive purifications of the Aro+ transductants on glucose M63 minimal plates, isolated colonies were tested for their maltose phenotype on maltose tetrazolium plates. The perA phenotype was tested by inoculating isolated colonies into MOPS medium containing 5 mM glucose, 5 x 10' M potassium phosphate, and 2 x 106 M ZnCl2. After 36 h of incubation (to induce phosphate deprivation), a portion of each culture was removed and assayed for alkaline phosphatase activity. a

236

WANNER., SART'HY, ANI) BECKWIT'H

TABLE

Bacteriophage P1 trans(luction of the

7.

perA allele"

ompB+"

ompBJ

perA+'

6 254

139 0

"(Recipient, phoR aroB perA+ ompB; donor, aroB+ perA ompB+; selected, aro+ (399). E. coli BW497 was transduced to prototrophy with a bacteriophage P1 lysate grown on BW489.2. Aro+ transductants were selected and purified on glucose M63 minimal plates before other phenotypes were tested. "The OmpB+ and OmpB - phenotypes were distinguished by stabbing a purified colony into a tryptoneveast extract plate which contained a soft agar overlay containing 2 x 10"' hy-2 phage per ml. On these plates only OmpB strains grew. The PerA+ and PerAJ phenotypes were distinguished by streaking a purified colony onto a /,-glycerol phosphate MOPS mininmal plate. Onlv 1'erA' strains yielded significant growth on these plates within 72 h.

TABI,F

8.

Bacteriophage P1 transduction of the ompB allele"

tTnselected marker

perA' perA

ornpB 5 131

decreased by the perA allele, which is repressed in the phoR + strains.

Unselected marker perA

*J. B3ACTERI()L.

urtlpI3 $369 3

Effect of perA allele on membrane proteins. Inner and outer membrane proteins were purified by the method of Diedrich et al. (10). The extracts were analyzed by electrophoresis on several gel systems. In Fig. 3, differences dependent upon the perA allele are observed in the gel pattern from the outer membrane samples. However, no detectable differences were observed in the gel pattern from the inner membrane samples on several different gel systems. With this gel system (26), it is clear that three outer membrane proteins are dramatically reduced by the perA allele. (Compare Fig. 3, lane c, with lanes a and g. See arrow heads on left.) For comparison the outer membrane preparation for an ompB strain was subjected to electrophoresis in lane b. Two bands are missing in the outer membranes from the ompB strain (see arrow heads on right). These correspond to proteins lb (uppermost band) and Ia (lower arrow head on right). The perA mutant, which has reduced levels of protein Ia, has simultaneously increased the level of protein lb. The increase in protein lb is probably a regulatory response which results when protein la is decreased (37).

Recipient, phoR aroB perA ompB+; donor, aroB+ perA+ ompB; selected, aro+ (505). E. coli BW496 was tranduced to prototrophy with a bacteriopohage P1 lysate grown on BW499. Aro+ transductants were selected and purified on glucose M63 minimal plates before testing. The OmpB and PerA phenotypes were tested as indicated in Table 7.

PerA+ or OmpB+ without any deliberate selection (data not shown). Effect of perA allele on other phosphate-

regulated proteins. During phosphate deprivation of a wild-type strain, at least three new proteins appear in the periplasm (23, 41; data not shown). Two of these correspond to alkaline phosphatase and a phosphate-binding protein, the phoS gene product. To examine the effect of the perA allele on the synthesis of the other phosphate-regulated proteins, we constructed isogenic strains that were phoR + orphoR and also aroB. Using bacteriophage P1 grown on a perA strain, we transduced these strains to prototrophy and then screened PerA+ and PerA transductants. The gel patterns for one set of otherwise isogenic strains are shown in Fig. 1. In this gel system, it can be seen that seven periplasmic proteins are decreased by the perA allele, as indicated by arrow heads on the left. Three periplasmic proteins are repressed by the phoR + allele, as indicated by arrow heads on the right. Alkaline phosphatase (lane c) is the only band

FIG. 3. The outer membranes were purified and subjected to gel electrophoretic analysis. The sepa-ating gel was 30 cm in length and it contained 9% acrylamide, 0.21"'. bisacrylamide, 0.1%7- SDS, and 8 M turea. A 5-cm section of the gel is illustrated. A portion of the outer membrane preparation representing 2 x 10" cells weas subjected to electrophoresits in each lane. The gel was stained u.ith Coomassie blue. Lane a, strain B W500.61 (per' omp+); lane b, strain B W500.1O (per+ ompB); lane c, strain B W500.1 (perA amp+); lanes d, e, and f, strains 503.425, 420, and 230, respectively (perA omp); and lane g, strain 503.10

(per+ omp+).

VOL. 140, 1979

MUTATION AFFECTING SECRETED PROTEINS

237

All three outer membrane proteins affected tion mechanism, thus reducing export of alkaline by perA appear in normal amounts in extracts phosphatase to the periplasmic space. This inpurified from a spontaneous revertant (data not terpretation is supported by the finding that at shown). In Fig. 3, lanes d, e, and f, outer mem- least 10 secreted proteins of the periplasm and brane extracts from the three perA ompB trans- the outer membrane are also reduced in amounts ductants have been analyzed. In each lane, four in the perA mutant. In addition to alkaline phosof the five outer membrane bands are greatly phatase, outer membrane protein Ia has been reduced. We attribute our finding of five, as identified as one of the proteins affected. If the opposed to four major outer membrane proteins perA mutant is defective in secretion, we might seen by others (37), either to improved resolu- have expected to find large amounts of precurtion or to a strain difference. sors of these secreted proteins present in the cytoplasm of the mutant strain. However, we DISCUSSION found no immunologically cross-reacting mateIn this paper, we describe a mutation, perA, rial to alkaline phosphatase in the cytoplasm, which results in a substantial reduction of the nor does the gel pattern of intracellular amounts of alkaline phosphatase in the E. coli appear to have been altered. This mayproteins not be periplasm. At the same time, strains carrying surprising, since it has been shown that precurthe perA mutation have reduced amounts of sors of secreted proteins which accumulate in several other periplasmic proteins and three the cytoplasm can be rapidly broken down (2). outer membrane proteins. Alternatively, if secretion and synthesis are couThe expression of alkaline phosphatase by the pled, inhibition of secretion may result in rephoA gene involves several steps. These include: duced synthesis. (i) interaction of RNA polymerase and Although the perA mutation does have pleioeffector(s) of the phoA gene at the promoter- tropic effects, it alters the synthesis of only a regulatory region to permit its transcription into portion of the periplasmic and outer membrane mRNA; (ii) translation of the mRNA into inac- proteins. For instance, whereas alkaline phostive protein monomers which are larger than the phatase is reduced in amounts, the phosphatemonomers isolated from mature enzyme (15); binding protein is present at the same levels in (iii) transport of the monomers across the inner perA and wild-type strains. Yet both proteins cell membrane during translation (36); (iv) oxi- are regulated by the same control system. Simdation of the sulfhydryl groups to form intra- ilarly, protein Ia of the outer membrane is missmolecular disulfide bridges (27); (v) aggregation ing, while protein lb is present in increased of the monomers and binding with zinc to form amounts. It is known that, although nearly all active dimers (34); and (vi) enzymatic removal envelope proteins analyzed do have signal seof a piece of the polypeptide chain from each quences, there are differences in at least certain monomer by a protease present in the mem- steps of the secretion process between them. For brane fractions (15). The temporal sequence of instance, the bacteriophage X receptor protein is some of these steps is unknown. The perA mu- secreted at the cell septum (28), whereas tant, which is defective in the synthesis of active Ia is exported at sites around the cell protein surface alkaline phosphatase, might have a mutation (35). Thus, the finding that the perA mutation affecting any of these several steps involved in affects only certain envelope proteins may indiphoA gene expression. cate that it has altered a component of the The results from several experiments demon- secretion mechanism for a subclass of envelope strate that the perA mutant does not affect proteins. We are currently examining the effect transcription initiation of the phoA gene. These of perA on other known proteins, such as the include the observations that: (i) the lower levels bacteriophage X receptor and the maltose-bindof alkaline phosphatase present in the perA mu- ing protein. If the perA mutation is defective in tant are regulated normally; (ii) the effect of the the secretion mechanism, then it might, for experA allele is unchanged in a mutant in which ample, affect a protein in the cytoplasmic memthe promoter-regulator region of the phoA gene brane which recognizes certain classes of has been altered; and (iii) the perA allele does sequences. Clearly, there is a variety of signal other not affect the level of phoA gene mRNA initia- explanations for the perA mutation in terms of tion, as judged by the lack of any effect of the the secretion mechanism. perA allele on 8)-galactosidase synthesis in a A second explanation for the phenotype of the phoA-lacZ fusion strain. perA mutant is that there is increased proteoThere exist several explanations for the phe- lytic activity in the bacterial envelope, which notype of the perA mutant. First, the mutation results in the degradation of certain susceptible may alter some component of the E. coli secre- periplasmic proteins. It would be somewhat sur-

238

WANNER, SART'HY, AND BECKWIH'H

tJ. BACTERIOL,.

prising if this were the case, since alkaline phos- described by Verhoef et al. Maps in the same region phatase is a protein unusually resistant to pro- as perA. teolytic enzymes. Finally, the perA gene may LITERATURE CITED code for a regulatory protein which acts to conB. J., K. B. Low, and A. L. Taylor. 1976. l. Bachmann, trol the synthesis of a number of envelope proRecalibrated linkage map of Escher-ichia coli K-12. teins at the level of their translation from Bacteriol. Rev. 40:116-167. mRNA. 2. Bassford, P., and J. Beckwith. 1979. Mutants of Escherichia coli which accumulate the precursor of a seCuriously, the perA mutation is closely linked creted protein in the cytoplasm. Nature (London) 277: to the ompB locus, which is involved in the 503-541. synthesis of outer membrane proteins Ia and lb. :3. Beacham, I. R., D. Haas, and E. Yagil. 1977. Mutants of Escherichia coli "cryptic" for certain periplasmic However, the ompB and perA strains have disenzymes: evidence for an alteration of the outer memtinct phenotypes, and ompB strains do not affect brane. ,. Bacteriol. 129:1034-1044. the synthesis of alkaline phosphatase. In one 4. Beckwith, J., T. Silhavy, H. Inouye, H. Shuman, M. case, it was shown that an ompB mutation alSchwartz, S. Emr, P. Bassford, and E. Brickman. tered the levels of alkaline phosphatase as as1978. The mechanisms of localization of proteins in Escherichia coli, p. 299-314. In S. C. Silverstein (ed.), sayed in whole cells, but this was shown to be Transport of macromolecules in cellular systems. Dahrelated to access of substrate to the enzyme, and lemkonferenzen, Berlin. no difference in amounts of enzyme was found 5. Beckwith, J. R., E. R. Signer, and W. Epstein. 1966. (3). Also, we have constructed a double perA Transposition of the lac region of E. coli. Cold Spring Harbor Symp. Quant. Biol. 31:393-401. ompB mutant which has the predicted phenoG., and B. Dobberstein. 1975. Transfer of protype for independent effects of the two muta- 6i. Blobel, teins across membranes. I. Presence of proteolytically tions. We believe that the two mutations reside processed and unprocessed nascent immunoglobulin in distinct genes. light chains on membrane-bound ribosomes of murine myelomia. J. Cell Biol. 67:835-851. In summary, we have isolated a pleiotropic E., and J. Beckwith. 1975. Analysis of the mutant which simultaneously affects the expres- 7. Brickman, regulation of Escherichia co/i alkaline phosphatase sion of 10 secreted proteins in E. coli. Substansubunits using deletions and ,80 transducing phages. J. tial evidence has been presented which demonMol. Biol. 96:307-316. strates that, at least for alkaline phosphatase, 8. Cancedda, R., and M. H. Schlesinger. 1974. Localization of polyribosomes containing alkaline phosphatase the perA allele affects some posttranscription nascent polypeptides on membranes of Escherichia initiation step in phoA gene expression. Addicoli. J. Bacteriol. 117:290-301. tional experiments are now in progress to deter- 9. Casadaban, M. 1976. Transposition and fusion of the lac genes to selected promoters in Escherichia coli using mine whether alkaline phosphatase monomers bacteriophages lambda and mu. J. Mol. Biol. 104:541are synthesized and rapidly degraded or whether 555. monomers are not synthesized. We also hope to 10. Diedrich, D. L., A. 0. Summers, and C. A. Schnaitidentify some of the other proteins affected by man. 1977. Outer membrane proteins of Escherichia coli. V. Evidence that protein la and bacteriophagethe perA allele. Further studies on this mutant directed protein 2 are different polypeptides. J. Bacteand others should be useful in unraveling the riol. 131:598-607. molecular basis of synthesis and secretion of 11. Dvorak, H. F., R. W. Brockman, and L. A. Heppel. alkaline phosphatase. 1967. Purification and properties of two acid phosphaACKNOWLEDGMENTS This work was supported by a grant from the American Cancer Society to J. R. Beckwith (VC-13F,G,H). Barry Wanner was supported as a postdoctoral fellow by a National Research Service Award (5F32 GM05456). We are grateful to B. Bachmann, E. Brickman, F. -Jacob, I. Guarente, A. Pugslev, and A. Torriani for providing hacterial strains. We thank R. MacGillivrav and A. McIntosh for tech-

12.

13.

14.

nical assistance. ADDENDUM We have recently learned that miutanits with similar properties to those described in this paper have been independently isolate(d by C. Wandersman, F. Moreno, and M. Schwartz (manuscript in preparation). These mutants were selected as resistant to phage TPI, a phage which can use either protein la or the lamB protein as its receptor.

ADDENDUM IN PROOF Verhoef et al. (Mol. Gen. Genet. 169:137-146, 1979) have recently published a paper describing mutants missing protein Ia btit Iot lb. The genetic lesion

15.

16.

1,.

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